Cooperative ligand binding to hemoglobin. Effects of temperature and pH on a hemoglobin with spectrophotometrically distinct chains (Tunnus thynnus).

Binding of CO to the hemoglobin of Tunnus thynnus has been studied by kinetic and static methods in the range pH 6.75-8.0 and at temperatures between 1.5-40 degrees C. The results may be described in terms of the two-state model of cooperativity (Monod. J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) extended to include chain differences. The rate constants for CO binding to alpha and beta chains in the R state, at 20 degrees C, were 8.4 and 1.9 microM-1 s-1 with associated apparent activation energies of 3.5 and 10.5 kcal mol-1. Dissociation from the R state was monophasic with a rate constant, at 20 degrees C, of 0.016 s-1 and Ea of 26.4 kcal mol-1. The difference spectrum for binding of CO to the alpha chain in the T state is displaced 2 nm to the red as compared to the spectra associated with CO binding to the beta chain in the T state and to both chains in the R state. The proportion of T state hemoglobin present at different fractional saturations can thus be measured and compared with the results predicted from the model. When the temperature is raised, the value of L decreases greatly with an enthalpy of +80 kcal mol-1. The effects of the change in L override the decrease in intrinsic CO affinity of T. thynnus hemoglobin in the R and T states and result in a reverse temperature dependence of CO equilibria compared to mammalian hemoglobins.